Characterization of rabbit liver P450IIE1 synthesized in transformed yeast cells.
cDNA for chimeric protein, P450(3P4), consisting of the amino-terminal 43 residues (the membrane-anchor region) of rabbit P450IIC14 and the remaining 447 residues of rabbit P450IIE1 was constructed, then cloned into expression vector pAAH5, and expressed in Saccharomyces cerevisiae AH22 cells under the control of yeast ADH1 promoter. P450(3P4) thus synthesized in the transformed yeast cells was partially purified, and its spectral and catalytic properties were examined. In the oxidized state P450(3P4) exhibited a high-spin type absorption spectrum even in the absence of a substrate. The reduced CO complex of the P450 showed a Soret absorption maximum at 452 nm. P450(3P4) catalyzed aniline p-hydroxylation, N-nitrosodimethylamine demethylation, benzphetamine N-demethylation, and laurate and caprate (omega-1)-hydroxylation in the reconstituted system containing the P450 and NADPH-P450 reductase. These results indicate that P450(3P4) preparation obtained from the transformed yeast cells has spectral and catalytic characteristics identical with those of P450IIE1 purified from rabbit liver microsomes, confirming the substrate specificity reported of P450IIE1.
Imai
Y
Y
Department of Veterinary Science, University of Osaka Prefecture.
Uno
T
T
Nakamura
M
M
D000818:Animals; D001483:Base Sequence; D019392:Cytochrome P-450 CYP2E1; D006900:Hydroxylation; D008745:Methylation; D008862:Microsomes, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D010089:Oxidoreductases, N-Demethylating / Q000096:biosynthesis / Q000235:genetics*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011817:Rabbits; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D013379:Substrate Specificity; D014170:Transformation, Genetic
D019392:Cytochrome P-450 CYP2E1; D010089:Oxidoreductases, N-Demethylating